1. Field of the Invention
The invention pertains to the utilization of systems and methods deploying the cyclic processes of vitrification for the expressed purposes of placing viable biological systems into a state of cryogenic suspension, the subsequent retrieval of the same said viable biological systems from the state of cryogenic suspension and their reintroduction to ambient conditions of temperature and pressure.
2. Description of the Prior Art
Conventional means of cryogenic preservation have exhibited great promise in sustaining unicellular organisms such as sperm, erythrocytes, various protozoa and multicellular embryonic or fetal systems, detached organs and the like. Significant drawbacks have occurred in attempts to place entire organ systems in a protracted state of cryogenic suspension due to a series of complications arising from intracellular and extracellular damage to chemical and structural biological systems attributed to the formations of crystals at the nucleation temperature (T.sub.n) and devitrification. The inadvertant extremes in baropressure and thermal parameters produced by cryogenic suspension often initiates the deactivation of labile enzymes and metabolites, a direct consequence of denaturization. Another potentially lethal consequence of cryogenics are the induced losses in ionic transport mechanism, imbalances in osmotic concentrations and high levels of toxicity due to the accumulation of cryoprotectants and the like compounds.
The aforementioned deficiencies in conventional cryogenic techniques are made evident in the precedings of Farrant (Nature, 205, 1284-87 1965), Fahy and Mac Farlane (20th annular Meeting of the Society of Cryobiology U.K. August 1983 supported by grants GM17959 and BSRG 2 507 RPOS737 NIH American Red Cross), Pierre Boutron (Cryobiology 21, 183, 191 (1984)). The utilization of cryoprotectants with inherent toxicological effects such as dimethyl sulfoxide, devitrification, extremes in temperature, pressure and the complications associated with incurred losses of viable chemicals, ionic transport and or the deactivation of essential enzymes or labile cofactors, which are evident in present techniques of cryogenic preservation. Thus, in recent years there has been a substantial interest in, and the development of effective viable alternatives to the foregoing conventional cryogenic techniques.
Accordingly, it is desirable to provide a method for the successful preservation of organs, tissues and other biological materials at very low temperatures which avoids the formation of ice crystals, minimizes the effective concentration of potentially harmful chemicals; and permits the rapid introduction and removal of cryoprotectants at feasible temperatures, without the necessity of elaborate equipment to monitor precise conditions of concentration and temperature. These advantages are obtained by the vitrification process of the present invention.
The principles of vitrification are well-known. Very generally, the lowest temperature a solution can be supercooled without freezing is the homogeneous nucleation temperature T.sub.n, at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution. Vitrifiable solutions have a glass transition temperature T.sub.g, higher than T.sub.n, at which temperature the solution vitrifies, or becomes non-crystalline solid. Owing to the kinetics of nucleation and crystal growth, it is effectively impossible for water molecules to align for crystal formation at temperatures much below T.sub.g.
On cooling most dilute aqueous solutions to the vitrification temperature (about -135.degree. C.), T.sub.n is encountered before T.sub.g, and ice nucleation occurs, which makes it impossible to vitrify the solution. In order to make such solutions useful in the preservation of biological materials by vitrification it is therefore necessary to change the properties of the solution so that vitrification occurs instead of ice crystal nucleation and growth. While it is known that many solutes, such as commonly employed cryoprotectants like dimethyl sulfoxide (DMSO) raise T.sub.g and lower T.sub.n, solution concentrations of DMSO or similar solutes high enough to permit vitrification typically approach the eutectic concentration and are generally toxic to biological material; further, careful development of such concentrations is necessary to avoid ice crystal formation. While it is also generally known that high hydrostatic pressures similarly raise T.sub.g and lower T.sub.n, vitrification of most dilute solutions by the application of pressure is either impossible or impractical. Further, for many solutions vitrifiable by the application of pressure, the required pressures cause unacceptably severe injury to unprotected biomaterials during vitrification thereof; for example, a pressure of only 1000 atm. is lethal to unprotected kidney slices. These and other barriers to cryopreservation of biological materials have not been surmounted in the prior art.